Exploring aromatic components differences and composition regularity of 5 kinds of these 4 aroma types Phoenix Dancong tea based on GC–MS

Different aromatic components do indeed give different tea flavors. There is still little research on whether there is a certain regularity in the combination and content of aromatic components in different aroma types of Phoenix Dancong (PDC) tea. This potential regularity may be a key factor in unraveling the relationship between reproduction and evolution in PDC tea. Here, the 5 kinds of these 4 aroma types PDC tea (Zhuye, Tuofu, Jianghuaxiang, Juduo, Yashixiang) were used as research materials in this study, the headspace solid-phase microextraction combined with gas chromatography–mass spectrometry was used to analyze the aromatic components of these PDC teas. The results showed a total of 36 aromatic components identified in this study. When conducting cluster analysis, it was found that similarity degree arrangement sequence of 5 PDC teas was Juduo, Tuofu, Yashixiang, Zhuye and Jianghuaxiang. Among these aromatic components, the 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione, the 2-Cyclopenten-1-one, 3-methyl-2-(2-pentenyl)-,(Z)-, the 2,4-Di-tert-butylphenol, the 3,7-dimethyl-1,5,7-Octatrien-3-ol, and the 2-Furanmethanol,5-ethenyltetrahydro-.alpha.,.alpha.,5-trimethyl-,cis- are common to 5 PDC teas. This study aims to elucidate the similarities in the aromatic components of 5 PDC teas, revealing the major aroma-endowed substances of various aroma, and providing theoretical reference for further exploring the relationship between aroma type discrimination, variety selection, and evolution of PDC teas.

on key aromatic compounds in tea has attracted much attention [7][8][9][10] .However, due to the relatively low content and complex types of aromatic compounds in tea, there have been few reports on the composition of aromatic components in different aroma types of PDC teas.There is even less research on the similarity or correlation between different types of PDC tea and different varieties of tea with the same aroma, which poses challenges to the development, selection of varieties, and study of evolutionary relationships in PDC tea.At present, the aromatic chemical species in aromatic components are commonly obtained by headspace solid-phase microextraction (HS-SPME) method 6,11,12 and gas chromatography-mass spectrometry (GC-MS) is used to analyze and identify the various aromatic compounds in tea [13][14][15] .Due to the complexity and diversity of tea aromatic components identified by GC-MS, corresponding methods must be combined to analyze and interpret them, such as principal component analysis (PCA) and hierarchical clustering analysis (HCA) methods 16,17 , which are used to evaluate various tea varieties and are an important tool for determining the quality of tea and the differences in aromatic components across regions [18][19][20] .Therefore, focusing on the purpose of this study, these analytical methods can be used to uncover the key aromatic substances between different tea varieties, and to study the similarity or correlation between different aroma types of tea and the same aroma type with different varieties tea.
Previous studies have evaluated the aromatic components of different aroma types of PDC tea 2,4,21 , but the studies were not specific to that variety and key aromatic components.In addition, there is a lack of corresponding research reports on the systematic comparison and classification of aromatic components in different aroma types of PDC tea, which is not conducive to studying the differences between PDC tea varieties and identifying those that belong to the corresponding aroma types of PDC teas.Therefore, in this study, the HS-SPME combined with GC-MS was used to extract and identify the aromatic components of 5 PDC teas of these 4 aroma types, PCA and hierarchical cluster analysis methods were used in combination with visualization using Venn diagrams to present the similarities and differences of the aromatic components of 5 PDC teas.Together, these were used to reveal the typical aroma features and core aroma-enhancing substances, as well as the combination and content patterns of different aroma types of PDC tea.The results show the similarities and differences of the aromatic components of 5 PDC teas, revealing their typical aroma characteristics, the core aroma-endowed substances, and the regularity of the combination and contents of the aromatic components of the different aroma types of PDC teas, thus providing a scientific basis for the development of PDC tea, the identification of aroma types, the selection and breeding of varieties, and the study of evolutionary relations.

Hierarchical cluster analysis of aromatic composition of 5 PDC teas
Hierarchical cluster analysis, which is mostly used to analyze multivariate data, is a multivariate statistical method that gradually aggregates samples based on the similarity or proximity of their quality features.Currently, it is widely used to distinguish tea varieties and identify tea quality 22 .For example, Wang 23 used hierarchical cluster analysis to distinguish black teas from 7 districts in Guangdong Province; Liu 6 characterized the flavor profiles of 20 compounds in 16 green tea samples from different geographic origins; Wang 24 used hierarchical cluster analysis to clearly distinguish two types of oolong tea based on their colors.In this study, the distribution of the 36 aromatic components in 5 PDC teas was also analyzed using hierarchical cluster analysis.The heat map clearly shows that these 40 aromatic components are mainly divided into 3 categories (Fig. 3).The first category is the Linalool, the Benzyl nitrate, the 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione, the 2,4-Di-tert-butylphenol, the 3,7-dimethyl-1,5,7-Octatrien-3-ol, the 2-Cyclopenten-1-one, and the 2-Furanethanol,5-ethyltetrahydro-.www.nature.com/scientificreports/These aromatic components differed comparatively among these 5 kinds of PDC tea, with the greatest differences being in the remaining 15 aromatic components of the third category.Differences in the types and relative contents of aroma components are the material basis for the differences in the quality of aroma.Combined with the hierarchical clustering analysis, the 1st category is containing the aroma components common to these 5 kinds of PDC teas, which may then be the aroma components that most PDC tea varieties have, retaining the aromatic traits of the parent plant before improvement.The most significant differences in aroma components among these 5 kinds of PDC teas are clustered in the 2nd and 3rd categories, indicating that the improvement of the variety has led to changes in the relative content and composition of internal substances in the tea, which ultimately result in differences in the taste of the tea soup.Therefore, this study can confirm that the differences in the aroma composition of these 5 kinds of PDC tea are all concentrated in the 2nd and 3rd categories, which may also be the root cause of the differences in these teas.Of course, in order to comprehensively analyze the differences between these teas, we have included in the subsequent discussion and analysis a discussion of the relevance of the results to other analyses.

Combination differences and composition regularity of aromatic components of 5 PDC teas
After classifying the 36 identified aromatic components, Tuofu was found to have the least variety of aromatic components, followed by Yashixiang and Juduo, the highest is Zhuye (Table 1).The relative content of alcohols in Zhuye was the highest (52.13%), followed by ketones (12.90%) and heterocycles (11.45%), and lowest for nitrogenous (2.18%).The relative content of alcohols in Tuofu was the highest (60.16%), followed by ketones (20.47%), then lactones (10.86%) and phenols (8.51%).The relative content of alcohols in Jianghuaxiang was the highest (62.41%), followed by heterocycles (13.11%) and ketones (9.88%) and the lowest was aldehydes (1.58%).The relative content of alcohols in Juduo was the highest (31.88%) followed by lactones (18.41%) and nitrogens (16.08%) and the least was aldehydes (2.58%).The relative content of ketones in Yashixiang was the highest (35.87%), followed by alcohols (26.84%) and heterocycles (20.93%), and the least were phenols (2.58%) and nitrogens (2.62%).In addition, this study also found that alcohols, ketones and phenols were common to these 5 PDC teas, and alcohols accounted for the largest relative content, followed by ketones, and the smallest was phenols (Table 2 and Fig. 5), which suggests that in terms of the relative content of alcohols and ketones dominated the aroma substances of PDC tea.Alcohols identified in the 5 PDC teas, Zhuye, Tuofu, Jianghuaxiang, Juduo, and Yashixiang, accounted for 52.13%, 60.16%, 62.41%, 31.88%, and 26.84% of their total content, respectively.Alcohols is an important aromatic-active compounds.Studies have shown that linalool, benzyl alcohol and geraniol are the main alcohol in green tea 6,25 .Unlike green tea, the main alcohol in this study is the 3,7-dimethyl-1,5,7-octatrien-3-ol (27.7%, 30.6%, 19.9%, 14.8% and 22.8%), and of course, linalool also played a role.In addition, the relative content of alcohol in Zhuye, Tuofu and Jianghuaxiang is significantly higher than Juduo and Yashixiang, which means that the aromatic odor provided by alcohol is the key source and typical aroma characteristics of the aroma of Zhuye, Tuofu and Jianghuaxiang, while 3,7-dimethyl-1,5,7-octatrien-3-ol is their core aroma substance.
In addition, ketones were another type key aromatic active compound in the 5 PDC teas.Ketones identified in Zhuye, Tuofu, Jianghuaxiang, Juduo and Yashixiang accounted for 12.90%, 20.47%, 9.88%, 9.30% and 35.87% of the total, respectively.One of the shared ketones substances is 2-Cyclopenten-1-one,3-methyl-2-(2-pentenyl)-,(Z)-, www.nature.com/scientificreports/which is the most abundant, and thus may be an important contributor to the flavor of these 5 PDC teas.Some studies have demonstrated that ketones contribute significantly to the aroma of tea because of their low overall odor threshold and high flavor dilution coefficient 6,26 .Ketones substance formation may be related to the degradation of unsaturated fatty acids and β-carotene within tea 27,28 .
In addition to abundant alcohol and ketones, phenols, aldehydes, lactones, heterocycles, siloxanes and nitrogenous also exist in these 5 PDC teas.It has been confirmed that phenols determine the bitter and astringent taste of tea 29 , and although the relative content of phenols is not high, they are present in all 5 PDC teas, implying that it is also a relatively important contributor to the flavor of PDC tea.The production of aldehydes substances in tea is mainly associated with the degradation of unsaturated fatty acids 30,31 .Aldehydes are considered to play an important role in the formation of aroma due to their extremely low odor threshold and similar odor characteristics 29,32 .In addition, lactones, heterocycles, siloxanes and nitrogenous are important aromatic components in tea.Lactones may give tea fruit flavor.Some heterocycles may be key substances in the formation of tea aroma.For example, indole has sweet taste and floral aroma, which can effectively improve the overall aroma of tea 33 .Siloxanes is also a key aroma component in tea, which appears to increase and then decrease during the preparation of tea samples 34 .Nitrogenous has a special nutty flavor, and its formation may be related to the production of amino acids and sugars 35,36 .However, this study found that aldehydes only existed in Zhuye, Jianghuaxiang and Juduo, while lactones did not exist in Jianghuaxiang.Heterocycles and nitrogenous are absent in Tuofu.Siloxanes only exist in Zhuye, Juduo and Yashixiang.Therefore, it is speculated that the distribution differences of these aroma types in 5 kinds of PDC tea may be the key factor of their flavor differences.It needs to be considered that our study did not use internal standards for quantitative analysis.Although we only wanted to explore the combination of aroma components, composition patterns, and core aroma substances of these four aroma types and five Phoenix Dancong teas.To be sure it is very important to quantify with internal standards, can we get similar conclusions if we have used internal standards for quantitative analysis?So, if we want to establish a complete method for identifying the varieties of Phoenix Dancong tea through differences in different aroma components, more experimental evidence is needed to prove and improve in the future.PCA is a non-target statistical method to categorize composite indicators according to a certain regular, which can highlight the similarities and differences between samples and sample content, and provide the objects contributing key components 6 .Studies have confirmed that PCA can reveal the similarities and differences between different teas according to the types and contents of aromatic components in tea 37,38 .PCA was used in this study to reflect the contribution of each variable to the 5 PDC teas.The first principal component (PC1) explained 53.9% of the variation and the second principal component (PC2) explained 19.5% of the variation of various aromatic components in the refined finished tea of 5 PDC teas (Fig. 6A).It is mainly contributed by the 3,7-dimethyl-1,5,7-Octatrien-3-ol, the 2-Cyclopenten-1-one, 3-methyl-2-(2-pentenyl)-, (z)-, the 2-furanmethanol,5-ethyltetrahydro-.alpha.,.alpha.,5-trimethyl-,cis-, the linalool, the indole and the Cyclopentasiloxane, decamethyl-.The first principal component (PC1) explained 66.4% of the variation and the second principal component (PC2) explained 16.4% of the variation in all types of aromatic components in the refined finished tea of 5 PDC teas (Fig. 6B), with the first principal component (PC1) being mainly contributed by alcohols and ketones.

Experimental materials
This study was conducted at Hanshan Normal University, Chaozhou City, Guangdong Province, China.Five different varieties of four aroma types PDC tea, Zhuye, Tuofu, Jianghuaxiang, Juduo, Yashixiang were used as the research objects.In the spring of 2022, fresh leaves of PDC tea with an average age of 10 years were taken from Fenghuang Mountain in the north of Fenghuang Town, Chaoan District, Chaozhou City, Guangdong Province using a multi-point sampling method.Fenghuang Mountain is located approximately 30 km from the urban area.The entire mountain is steep and upright, with its main peak at an altitude of 1497.8 m.The climate of this region belongs to the subtropical climate zone, with abundant sunshine and rainfall.The average temperature is about 21.4 °C, with the highest temperature recorded at 39.6 °C and the lowest temperature recorded at − 0.5 °C.
The average annual rainfall is 1685.8mm.It has fertile, acidic soil with abundant sunlight resources, which is extremely suitable for the growth of certain varieties of tea trees.Liu et al. 39 have described the source of PDC tea and the identification of different varieties.In addition, the PDC tea specimen information is not yet public and

Conclusions
In this study, a total of 40 major aromatic components were detected based on GC-MS of 5 kinds of these 4 aroma types PDC tea, including Zhuye, Tuofu, Jianghuaxiang, Juduo and Yashixiang identified 21, 10, 17, 16 and 14 aromatic components, respectively.And the 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione, the 2-Cyclopenten-1-one,3-methyl-2-(2-pentenyl)-,(Z)-, the 2,4-Di-tert-butylphenol, the 3,7-dimethyl-1,5,7-Octatrien-3-ol and 2-Furanmethanol,5-ethenyltetrahydro-.alpha.,.alpha.,5-trimethyl-,cis-thesearomatic components are common to these 5 kinds teas.The results of the PCA showed that alcohols and ketones were the main types of aromatic components of these 5 kinds tea, with a high contribution to the aroma of the tea, indicating that the composition and content of the aromatic components of these 5 PDC teas had a certain regularity, which included the differences in the content of the common components as well as the differences in the types of the unique aromatic components.Combined with the clustering heat map analysis, the similarity ranking sequence was Juduo, Tuofu, Yashixiang, Zhuye and Jianghuaxiang.These new findings clarified the differences of aromatic components, main types and similarity of aromatic components of the 5 kinds tea.The continuous development of tea cultivation technology, so that in the same aroma tea and evolved a variety of varieties of tea.However, the current assessment of different types of PDC tea varieties remain in the subjective awareness of the determination, the lack of scientific and effective uniform standards.This will lead to incorrect evaluation of a certain variety

Figure 3 .
Figure 3. Heat map of hierarchical clustering of different aromatic components of 5 kinds of these 4 aroma types PDC tea samples.

Figure 5 .
Figure 5.Comparison among volatile compounds in 5 kinds of these 4 aroma types PDC tea samples.All data are presented as mean ± standard error (SE, n = 3).Different letters above bars indicate statistical significance (P < 0.05).

Table 2 .
Relative contents of various aromatic components of 5 kinds of these 4 aroma types PDC tea samples.